Canine RANKL and methods for preparing and using the same

ABSTRACT

The present invention provides isolated nucleic acid molecules that encode a substantial part of canine RANKL polypeptide, including the extracellular domains of that polypeptide, the polypeptide and fragments thereof. Vectors and host cells encoding and expressing canine RANKL polypeptide are provided, as well as antibodies that bind to RANKL and that inhibit RANKL activity. Also provided are methods of treating an animal to inhibit or treat the loss of bone minerals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing of International ApplicationNo. US2003/039292, filed on Dec. 10, 2003 that claims priority under 35U.S.C. § 119(e) of provisional application U.S. Ser. No. 60/432,092filed Dec. 10, 2002, the contents of which are hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present application relates to nucleic acids encoding canine RANKligand (RANKL) polypeptides, canine RANKL polypeptides, immunogeniccompositions and/or vaccines comprising canine RANKL polypeptides,antagonists of canine RANKL, methods for identifying antagonists ofcanine RANKL, and methods for treating RANKL-mediated medicalconditions.

BACKGROUND OF THE INVENTION

Bone tissue is a composite of proteins, cells and minerals known as bonematrix. In a living animal, cells called osteoblasts build bone matrixand cells called osteoclasts break down and resorb bone matrix.Osteoblasts arise from mesenchymal stem cells and produce bone matrixduring development, after bone injury, and during the normal boneremodeling that occurs throughout life. Osteoclasts differentiate fromhematopoietic precursors of the monocyte-macrophage lineage and resorbbone matrix to support normal bone remodeling, in response to injury orstress, and in response to various disease states.

The equilibrium between the construction and resorption of bone matrixis regulated by numerous factors. One of the systems that regulates bonephysiology is the OPG/RANKL/RANK system. This system includes threefactors: osteoprotegerin (“OPG”); receptor activator of NF-κB (“RANK”);and RANK ligand “RANKL”).

RANK ligand, or RANKL, also variously art-known as ODF (osteoclastdifferentiation factor), OPGL (osteoprotegerin ligand) and TRANCE(TNF-related activation-induced cytokine), and by other designations, isa member of the TNF ligand family. RANKL exists in two forms: a cellularmembrane-bound form and a soluble form. RANKL mRNA exhibits its highestlevel of expression in bone. The major role of RANKL in bone is tostimulate osteoclast differentiation and activity, and to inhibitosteoclast apoptosis. In the presence of low levels of macrophage-colonystimulating factor (M-CSF), RANKL appears to be both necessary andsufficient for the complete differentiation of osteoclast precursorcells into mature osteoclasts. RANKL mRNA is also expressed in lymphoidtissues, such as the lymph node, thymus, spleen, fetal liver and Peyer'spatches. In addition, RANKL has a number of effects on immune cells.These effects include the activation of c-Jun N-terminal kinase (JNK) inT cells, inhibition of apoptosis of dendritic cells, induction ofcluster formation by dendritic cells and effects on cytokine-activated Tcell proliferation.

The RANKL/RANK signaling pathway has been characterized. RANKL,expressed on the surface of pre-osteoblast/stromal cells or in solubleform, binds to RANK, which is expressed on osteoclast precursor cells.This binding promotes the differentiation of osteoclast precursor cellsinto mature osteoclasts. Macrophage colony stimulating factor (M-CSF),which binds to its receptor, c-Fms, on preosteoclastic cells, appears tobe involved in osteoclast development because it is the primarydeterminant of the pools of these precursor cells. OPG is a solublereceptor for RANKL, and can block the effects of RANKL by acting as a“decoy” binding target. In addition, a number of cytokines, includingTNF-α and IL-1, modulate the system, for example, by stimulating M-CSFproduction or by increasing RANKL expression.

Proper functioning of the OPG/RANKL/RANK system is essential for bonemetabolism, immune functions and vascular functions. Disruptions in thissystem have been implicated in various skeletal and immune disorders,such as rheumatoid arthritis, osteoporosis and osteopetrosis.Antagonists of RANKL can be used to treat osteoporosis and otherconditions mediated by such RANKL/RANK interactions. Assays for theidentification of such antagonists to human, mouse and rat RANKL havebeen enabled by the isolation of human, mouse and rat RANKLpolypeptides. Because the antigenic (extracellular) domain of RANKLvaries among species, species specific RANKL polypeptides are preferredfor identifying species specific RANKL antagonists.

The RANKL proteins and encoding genes for several mammalian species areknown. For example, human RANKL protein and encoding nucleic acid isdescribed, for example, by U.S. Pat. No. 6,242,213. Rat RANKL proteinand encoding nucleic acid is described, for example, by Internationalpublished patent appl. No. WO 01/23549. Murine RANKL protein andencoding nucleic acid is described, for example, by co-owned U.S. Pat.No. 6,242,586. Methods of modulating the effects of RANKL from severalnon-canine sources are described, for example, by co-owned U.S. Pat. No.6,525,180, by 6,316,408, and by Halkier et al., in publishedinternational patent application (WO0015807A1, March 2000), However,there remains a longfelt and heretofore unmet need in the art for theidentification and production of canine RANKL, as well as for newmethods and antagonists for modulating the effects of canine RANKL incanine and other animal, e.g., mammalian species.

The citation of any reference herein should not be construed as anadmission that such reference is available as “prior art” to the instantapplication.

SUMMARY OF THE INVENTION

These and other problems are solved by the instant invention, thatprovides for nucleic acids encoding substantially all of the canineRANKL polypeptide, and methods of making and using the same. Inparticular, the present invention provides for an isolated nucleic acidmolecule, and its complement, that includes a nucleic acid sequenceencoding a polypeptide, wherein the encoded polypeptide includes aminoacid residues according to SEQ ID NO:2. The present invention alsoprovides a nucleic acid molecule that hybridizes to the complement ofthe isolated nucleic acid molecule under stringent conditions, providedthat the hybridizable nucleic acid molecule does not encode a human,murine or rat RANKL. The artisan will appreciate that the isolatednucleic acid molecule of the present invention is optionally DNA or RNA.

The present invention also provides an isolated canine RANKL polypeptideaccording to SEQ ID NO:2, or a fragment thereof, that binds to canineRANK. Fragments of the canine RANKL polypeptide include any antigenicfragments, and preferably those enumerated by Tables 1 and 2, shownherein, below.

The present invention further provides immunogenic compositions thatinclude the canine RANKL polypeptide, and the previously mentionedfragments thereof.

The present invention still further provides an immunogenic compositionthat includes the canine RANKL polypeptide, and/or an antigenic fragmentthereof, e.g., listed by Tables 1 and 2, herein, below. The immunogeniccomposition is preferably in a vaccine composition, optionally includinga suitable pharmaceutically acceptable carrier, i.e., comprisingisotonic saline, physiologically acceptable buffer(s) and an effectiveart-known adjuvant, as required. More preferably, the immunogeniccomposition also includes at least one additional element incorporatedinto an immunogenic composition and/or into a fusion protein or acovalent conjugate linking the additional element to the canine RANKLpolypeptide or fragment. The additional element can be at least one ofthe following and/or any combination thereof:

-   -   (a) at least one foreign T helper lymphocyte epitope,    -   (b) at least one element that targets the canine RANKL        immunogenic composition to an antigen presenting cell or a        B-lymphocyte,    -   (c) at least one element that stimulates the immune system,    -   (d) at least one element that optimizes presentation of the        canine RANKL to the immune system.

The present invention also provides a polyclonal or monoclonal antibodyor a functional fragment thereof that selectively binds to canine RANKL,and optionally RANKL of other animals, e.g., other mammals. Methods forinhibiting RANKL activity in an animal, e.g., a mammal, by administeringto the animal an amount of the antibody or fragment thereof that iseffective to inhibit RANKL activity in the mammal, are also provided.

The artisan will appreciate that the antibody is administered at afrequency and for a duration sufficient to maintain bone mass and/orbone density in the mammal at a level equal to or greater than the bonemass or bone density measured prior to the step of administering theantibody and/or fragment thereof.

In addition to employing the anti-RANKL antibodies of the presentinvention to treat or inhibit the loss of bone minerals in an animalsuch as a mammal, and particularly a canine, such antibodies can beelicited in situ by immunizing the animal to be treated with canineRANKL, or fragments thereof, with an immunogenic form of the canineRANKL. The present invention further provides methods for inhibitingRANKL activity in a mammal that comprise administering to the mammal anamount of a RANKL immunogenic composition capable of effectivelyeliciting antibodies that selectively bind to RANKL in the mammal. Sucha RANKL immunogenic composition can either comprise or consist of apolypeptide having the amino acid sequence of SEQ ID NO:2, or a fragmentthereof. The fragment of the polypeptide having the amino acid sequenceof SEQ ID NO:2 in this case would be both capable of binding the caninereceptor activator of NF-κB, and of effectively eliciting antibodiesthat selectively bind to RANKL in the mammal. Mammals that can betreated by direct administration of an antibody of the present inventionor its functional equivalent, and/or through immunization as providedabove, include, but are not limited to, a canine, an equine, a feline, abovine, a porcine, and a human.

Also provided are nucleic acids, either RNA or DNA, encoding canineRANKL and fragments thereof, e.g., the polypeptide fragments listed byTables 1 and 2, herein, below, replicable nucleic acid vectors, hostcells comprising the vectors, and methods of producing the inventivepolypeptide(s) by culturing the host cells under conditions suitable forexpression the polypeptide. The vector can be a plasmid, a phage, acosmid, a mini-chromosome, and a virus, suitable for prokaryiotic oreukaryiotic host cell (e.g., a bacterium, a yeast, a protozoan, afungus, an insect cell, a plant cell, and a mammalian cell).

In particular, the replicable vector includes a suitable promotoroperably linked 5′ to the open reading frame of the canine RANKLimmunogenic composition of interest. The present invention furtherprovides stable cell lines comprising a replicable vector of the presentinvention. Such cell lines can secrete and/or express on its surface, acanine RANKL polypeptide, a fragment thereof, a fusion proteincomprising the RANKL polypeptide or fragment thereof, or any otherexpressible canine RANKL immunogenic composition.

These and other aspects of the present invention will be betterappreciated by reference to the following Detailed Description.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides for nucleic acid moleculesencoding substantially all of the canine RANKL polypeptide, includingthe entire extracellular (antigenic) portion of the protein. The canineRANKL is a type II membrane protein having an N-terminal intracellulardomain (approximately 48 amino acids) followed by a transmembrane domain(amino acids 49-68) and an extracellular domain (amino acids 69-319).The nucleic acid sequence and the corresponding amino acid sequenceencoding the extracellular domain, transmembrane domain and a portion ofthe intracellular domain of the canine RANK ligand is defined by SEQ IDNO: 1 and SEQ ID NO: 2, respectively. Specifically, the nucleic acid andamino acid sequences for canine RANKL are complete, except for the first129 nucleotides of the nucleic acid and corresponding 43 amino acids ofthe polypeptide.

The nucleic acid molecules of the present invention were isolated froman activated canine splenocyte cDNA library. The library may either beobtained commercially, or constructed according to methods known in theart. Simply by way of example, a suitable cDNA library is optionallyconstructed by producing 3W Th1 or Th2 cells as described, e.g., inOpenshaw, et al. (1995) J. Exp. Med. 182:1357-1367, incorporated byreference herein in its entirety. Briefly, Th1 or Th2 populations arederived from canine CD4+ T cells stimulated with antigen and antigenpresenting cells in the presence of IL-12 or IL-4. Cells are stimulatedonce each week for 3 weeks, then harvested and restimulated, e.g., withPMA and ionomycin for 4 h. See, Murphy, et al. (1996) J. Exp. Med. 183:901-913. Preferably, the cDNA library is prepared by the method of Bolinet al., (1997), The Journal of Neuroscience, 17(14):5493-5502,incorporated by reference herein its entirety.

Total RNA is isolated from the harvested cells using standard methodsknown in the art, e.g., using the guanidine thiocyanate/CsCl gradientprocedure as described by Chirgwin, et al. (1978) Biochem. 18:5294-5299.Poly(A) +RNA is isolated using, e.g., the OLIGOTEX mRNA isolation kit(QIAGEN). RNA from these cells is used to synthesize first strand cDNA,e.g., by using NotI/Oligo-dT primer (Gibco-BRL, Gaithersburg, Md.).Double-stranded cDNA is synthesized, ligated with BstXI adaptors,digested with NotI, size fractionated for >0.5 kilobase pairs (kb) andligated into the NotI/BstXI sites of pJFE-14, a derivative of the pCDSRαvector. See, for instance, Takebe, et al. (1985) Mol. Cell Biol.8:466-472. Electro-competent E. coli DH10α cells (Gibco-BRL) are usedfor transformation.

Canine RANKL was therefore cloned from a canine splenocyte cDNA libraryby employing a strategy of conducting a series of nested PCR reactions.Nested PCR involves two sequential PCR reactions, where the firstreaction product provides guidance for designing the primers for thenext PCR reaction. Each PCR reaction described in Example 1, below,generally contained 0.02 μg/μl of nucleic acid template, 1X PCR buffer,0.8 mM dNTP's, 1.1 mM Mg(OAC)₂, 0.16 units/μl of rTth polymerase(recombinant thermostable Taq polymerase), 2 OD/ml of vector primer, and0.2 OD/ml of gene specific primer. The nested PCR was performed,starting with the canine splenocyte activated cDNA library, using aGeneAmp XL PCR kit (Perkin Elmer, Branchburg, N.J.).

In the initial reaction, 30 cycles of PCR were performed using a vectorspecific primer and a gene specific primer (same or cross-speciesprimer). The PCR reaction was conducted with cycling between 94° C. (for1 minute) and 65° C. (for 5 minutes) for 30 cycles, followed by heatingto 72° C. for 10 minutes. Subsequently, a small aliquot of the PCRproduct of the first reaction served as the template for a second PCRreaction.

The second PCR reaction used gene specific primers (same or crossspecies) that hybridized to sequences internal to or nested between thefirst set of primers. This is called double nesting. The second PCRreaction was cycled according to the same protocol as the firstreaction. However, in some cases, the first reaction gene specificprimer was used in the second set of reactions with a different genespecific primer (same or cross species) for a single nesting reaction.

To provide a better appreciation of the present invention, the followingterms are defined.

As used herein, the terms “canine RANK ligand” and “canine RANKL” aredefined to mean any molecule capable of specifically binding to thecanine RANK receptor. Thus, the definition includes a canine RANKLpolypeptide that includes the peptide sequence defined by SEQ ID NO: 2or a portion thereof, while optionally avoiding 100% homology with otherart-known RANKL polypeptides isolated from non-canine mammalian species,e.g., optionally excluding the specific nucleic acid and/or polypeptidesequences encoding human RANKL, murine RANKL, rat RANKL, and/or anyother RANKL species that are presently art-known.

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is naturally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an isolated mRNA, cDNA or restriction fragment. In anotherembodiment, an isolated nucleic acid is preferably excised from thechromosome in which it may be found, and more preferably, is no longerjoined to non-regulatory, non-coding regions, or to other genes, locatedupstream or downstream of the gene contained by the isolated nucleicacid molecule when found in the chromosome. In yet another embodiment,the isolated nucleic acid lacks one or more introns, e.g., a cDNA.Isolated nucleic acid molecules are also contemplated to includesequences inserted into plasmids, cosmids, artificial chromosomes andthe like. Thus, in a specific embodiment, a recombinant nucleic acid isan isolated nucleic acid. In certain embodiments, it is useful to allowan isolated protein or nucleic acid to associate with other proteins ornucleic acids, or both, or with cellular membranes if it is amembrane-associated protein in order to achieve a desirable utility. Anisolated material may be, but need not be, purified.

The terms “purified” or “isolated” as employed herein refers tomaterials separated under conditions that reduce or eliminate thepresence of unrelated materials, i.e., contaminants, including nativematerials from which the material is obtained. For example, a purifiedor isolated protein is preferably free of other proteins or nucleicacids with which it can be found within a cell. A purified material maycontain less than about 50%, preferably less than about 75%, and mostpreferably less than about 90%, of the cellular components with which itwas originally associated. Purity can be evaluated by chromatography,gel electrophoresis, immunoassay, composition analysis, biological assayand other methods known in the art.

Methods for purification are well-known in the art. For example, nucleicacids can be purified by precipitation, chromatography,ultracentrifugation and other means. Polypeptides and proteins can bepurified by various methods including, without limitation, preparativedisc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phaseHPLC, gel filtration, ion exchange and partition chromatography,precipitation and salting-out chromatography, extraction andcountercurrent distribution. For some purposes, it is preferable toproduce the polypeptide in a recombinant system in which the proteincontains an additional sequence tag that facilitates purification, suchas, but not limited to, a polyhistidine sequence or a sequence thatspecifically binds to an antibody, such as FLAG and GST. The polypeptidecan then be purified from a crude lysate of the host cell bychromatography on an appropriate solid-phase matrix. Alternatively,antibodies produced against the protein or against a peptide derivedtherefrom can be used as purification reagents.

The term “substantially pure” indicates the highest degree of puritywhich can be achieved using conventional purification techniques knownin the art and means a canine RANKL polypeptide, nucleic acid or othermaterial that is free from other contaminating proteins, nucleic acidsand other biologicals derived from an original source organism orrecombinant DNA expression system. Substantial purity may be assayed bystandard methods and will typically exceed at least about 75%,preferably at least about 90%, more preferably at least about 95% andmost preferably at least about 99% purity. Purity evaluation may be madeon a mass or molar basis.

A “polypeptide” is a chain of amino acids that are linked together bypeptide bonds. Optionally, a polypeptide may lack certain amino acidresidues that are encoded by a gene or by an mRNA. For example, a geneor mRNA molecule may encode a sequence of amino acid residues on theN-terminus of a polypeptide (i.e., a signal sequence) that is cleavedfrom, and therefore, may not be part of the final protein.

Further the use of singular terms for convenience in description is inno way intended to be so limiting. Thus, for example, reference to acomposition comprising “an antibody” includes reference to one or moreof such antibodies. It is also to be understood that the presentinvention is not limited to the particular configurations, processsteps, and materials disclosed herein as such configurations, processsteps, and materials may vary somewhat. It is also to be understood thatthe terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims and equivalents thereof.

Preferably, a polypeptide according to the present invention is thecanine RANK ligand having an amino acid sequence defined by SEQ ID NO:2. Alternatively, a polypeptide comprises a subsequence of the aminoacid sequence defined by SEQ ID NO: 2 containing at least about 8,preferably at least about 12, more preferably at least about 20, andmost preferably at least about 30 or more contiguous amino acidresidues, up to and including the total number of residues in theligand. The polypeptides of the present invention can comprise any partof the sequence of such a ligand, and especially those fragments thathave heretofore not been known to the art. Polypeptides can be producedby proteolytic cleavage of an intact ligand, by chemical synthesis or bythe application of recombinant DNA technology. A polypeptide may benative or wild-type, meaning that it is identical to a polypeptide thatoccurs in nature; or it may be a mutein, a variant, an analog orotherwise modified, meaning that is has been made, altered, derived oris in some way different or changed from a native polypeptide.

The modifications that occur in a polypeptide are often a function ofhow the polypeptide is made. For canine RANK ligand polypeptides made byexpressing a cloned gene in a host, for instance, the nature and extentof the modifications will be determined in large part by the host cell'spost-translational modification capacity and the modification signalspresent in the amino acid sequence of the polypeptide. For example,glycosylation often does not occur in bacterial hosts, such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same post-translational glycosylations asmammalian cells; for this reason, insect cell expression systems havebeen developed to efficiently express mammalian proteins having theirnative patterns of glycosylation. Similar considerations apply to othermodifications. It will be appreciated that the same types ofmodifications may be present in the same or varying degrees at severalsites within a given polypeptide. Also, a given polypeptide may containmany types of modifications.

Varients of the subject canine RANKL polypeptide and encoding nucleicacid(s) have several utilities. In one embodiment, the canine RANKL ismodified to provide nonfunctional binding to the RANK binding-partner,resulting in a blocking of physiological response to canine RANKLpolypeptides. The inventive canine RANKL polypeptide and fragmentsthereof therefore provide agents for screening assays to identify RANKcompetitive or noncompetitive antagonists of canine or non-canine RANKfunction. Thus, in vitro assays of the present invention will often useisolated protein, membranes from cells expressing a membrane associatedrecombinant canine RANKL polypeptide, soluble fragments comprisingantigen binding segments of these proteins, or fragments attached tosolid phase substrates. These assays will also allow for the diagnosticdetermination of the effects of either binding segment mutations andmodifications, or antigen mutations and modifications, e.g., canineRANKL polypeptide analogs.

In an alternative embodiment, the inventive canine RANKL polypeptide andfragments thereof provide immunogens, i.e., the polypeptide and/orfragments thereof suitable for inducing a useful immune response in amammal so treated. The resulting immunity will serve to provide a sourcefor anti-canine RANK binding antibodies, anti-canine RANKL T-cells forgenerating useful mAb-producing hybridomas, and/or to downregulate RANKfunction in the treated mammal, thereby maintaining or enhancing bonedensity and/or bone strength in the treated mammal.

It should be noted that mammals exhibit tolerance to self proteins, thusadministration, i.e., vaccination of a mammal of the canine species withcanine RANKL polypeptide, by itself, without more, is not expected toprovide a useful level of immunization. Several additional strategiesare employed in order to present the inventive RANKL polypeptide to amammalian immune system in a way that results in an effective immuneresponse.

In one preferred embodiment, the inventive canine RANKL polypeptide isadministered with suitable adjuvants, in order to present thepolypeptide epitopes to the immune system in a way that is recognized as“foreign” or non-self. In another preferred embodiment, the inventivecanine RANKL polypeptide or fragments thereof are modified by anyart-known mutagenesis method to render it more antigenic to a caninespecies mammal. In a further preferred embodiment, all or part of theinventive RANKL polypeptide is joined by chemical synthetic or geneticengineering methods with one or more additional peptide domains to forman immunogenic fusion protein for administration to canines or othermammalian or avian species.

In yet a further embodiment, canine RANKL polypeptide is employed toelicit an immune response in a non-canine animal, including mammals, andparticularly humans. For example, human, mouse, rat and canine RANKLpolypeptide are not fully homologous. Thus, the canine RANKL polypeptideprovides a natural immunogen that is recognized as foreign, or non-self,by the equine, feline, bovine, porcine, and human immune systems forexample, without further modification or variation being required. Ofcourse, administration of canine RANKL polypeptide or fragments thereofto a non-canine mammal or to an avian (for enhancing egg production),will optionally be in combination with a suitable vaccine composition,including adjvants, and the like, as discussed in more detail herein,below.

For those embodiments of the present invention that comprise canineRANKL variants, the term “Variant(s)”, is used herein to describepolynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide respectively. The term variants includephysical variants, such as sequence variants and post-translationalvariants, and functional variants, such as analogs. Variants in thissense are described below and elsewhere in the present disclosure ingreater detail.

Glycosylation variants include, e.g., variants made by modifyingglycosylation patterns during synthesis and processing in variousalternative eukaryotic host expression systems, or during furtherprocessing steps. Particularly preferred methods for producingglycosylation modifications include exposing the canine RANK ligand toglycosylating enzymes derived from cells that normally carry out suchprocessing, such as mammalian glycosylation enzymes. Alternatively,deglycosylation enzymes can be used to remove carbohydrates attachedduring production in eukaryotic expression systems.

(1) A variant can be a polynucleotide that differs in nucleotidesequence from a reference polynucleotide. Changes in the nucleotidesequence of the variant may be silent, i.e., they do not alter the aminoacid sequence encoded by the polynucleotide. Where alterations arelimited to silent changes of this type, a variant will encode apolypeptide with the same amino acid sequence as the polypeptide encodedby the reference polynucleotide. However, changes in the nucleotidesequence of the variant may alter its amino acid sequence. Suchnucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below.

(2) Alternatively, a variant may be a polypeptide that differs in aminoacid sequence from a reference polypeptide. Generally, differences arelimited so that the amino acid sequences of the reference and thevariant are closely similar overall and, in many regions, identical.Variant and reference polypeptides may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

(3) A variant may also be a fragment of a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptide sequence bybeing shorter than the reference sequence, such as by a terminal orinternal deletion. A variant of a polypeptide also includes apolypeptide which retains essentially the same biological function oractivity as such polypeptide, e.g., pro-proteins which can be activatedby cleavage of the pro-protein portion to produce an active maturepolypeptide.

(4) A variant may also be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a pro-protein sequence.

(5) Furthermore, a variant of the polynucleotide or polypeptide may be anaturally occurring variant, such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the polynucleotide may be madeby mutagenesis techniques, including those applied to polynucleotides,cells or organisms, or may be made by recombinant means. Amongpolynucleotide variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

All such variants defined above in (1)-(5) are deemed to be within thescope of those skilled in the art, except that RANKL polypeptidevariants that are 100% homologous to human RANKL, murine RANKL, ratRANKL and/or any other heretofore art-known non-canine RANKLpolypeptides, and/or art-known non-canine RANKL-encoding nucleic acids,are preferably excluded from the scope of the present invention.

The present application also encompasses analogs of the canine RANKligand. The term “analog(s)” means a RANK ligand of the presentinvention which has been modified by deletion, addition, modification orsubstitution of one or more amino acid residues in the wild-type canineligand. It encompasses allelic and polymorphic variants, and alsomuteins and fusion proteins which comprise all or a significant part ofsuch canine RANK ligand, e.g., covalently linked via a side-chain groupor terminal residue to a different protein, polypeptide or moiety(fusion partner).

Some analogs are truncated variants in which residues have beensuccessively deleted from the amino- and/or carboxy-termini, whilesubstantially retaining the characteristic RANK binding activity.Substantial retention of binding activity by the foregoing analogs ofcanine RANKL typically entail retention of at least about 50%,preferably at least about 75%, more preferably at least about 80%, andmost preferably at least about 90% of the RANK binding activity and/orspecificity of the corresponding wild-type ligand.

Modifications of amino acid residues may include, but are not limitedto, aliphatic esters or amides of the carboxyl terminus or of residuescontaining carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino-terminalamino acid or amino-group containing residues, e.g., lysine or arginine.Other analogs are canine RANK ligands containing modifications, such asthe incorporation of unnatural amino acid residues or phosphorylatedamino acid residues, such as phosphotyrosine, phosphoserine orphosphothreonine residues. Other potential modifications includesulfonation, biotinylation, or the addition of other moieties.

Some amino acid substitutions are preferably conservative, with residuesreplaced with physically or chemically similar residues, such asGly/Ala, Asp/Glu, Val/Ile/Leu, Lys/Arg, Asn/Gln and Phe/Trp/Tyr. Analogshaving such conservative substitutions typically retain substantial RANKbinding activity. Other analogs, which have non-conservativesubstitutions, such as Asn/Glu, Val/Tyr and His/Glu, may substantiallylack such activity. Nevertheless, such non-conservative analogs areuseful because they can be used as antigens to elicit the production ofantibodies in an immunologically competent host. Because these analogsretain many of the epitopes (antigenic determinants) of the wild-typeligands from which they are derived, many antibodies produced againstthem can also bind to the active-conformation or denatured wild-typeligands. Accordingly, such antibodies can also be used, e.g., for theimmunopurification or immunoassay of the wild-type ligands.

Analogs of canine RANKL can be prepared by chemical synthesis or byusing site-directed mutagenesis [Gillman et al., Gene 8:81 (1979);Roberts et al., Nature, 328:731 (1987) or Innis (Ed.), 1990, PCRProtocols: A Guide to Methods and Applications, Academic Press, NewYork, N.Y.] or the polymerase chain reaction method [PCR; Saiki et al.,Science 239:487 (1988)], as exemplified by Daugherty et al. [NucleicAcids Res. 19:2471 (1991)] to modify nucleic acids encoding the ligand.Adding epitope tags for purification or detection of recombinantproducts is also envisioned. General techniques for nucleic acidmanipulation and expression that can be used to make the analogs aredescribed generally, e.g., in Sambrook, et al., Molecular Cloning: ALaboratory Manual (2d ed.), 1989, Vols. 1-3, Cold Spring HarborLaboratory. Techniques for the synthesis of polypeptides are described,for example, in Merrifield, J. Amer. Chem. Soc. 85:2149 (1963);Merrifield, Science 232:341 (1986); and Atherton et al., Solid PhasePeptide Synthesis: A Practical Approach, 1989, IRL Press, Oxford. Stillother analogs are prepared by the use of agents known in the art fortheir usefulness in cross-linking proteins through reactive side groups.Preferred derivatization sites with cross-linking agents are free aminogroups, carbohydrate moieties and cysteine residues. In an optionalembodiment, RANKL polypeptide analogs that are 100% homologous to humanRANKL, murine RANKL, rat RANKL and/or any other heretofore art-knownnon-canine RANKL polypeptides, and/or art-known non-canine RANKLencoding nucleic acids, are excluded from the scope of the presentinvention.

Preferred polypeptide fragments of SEQ ID NO:2 are antigenic, and willinduce a protective immunity in a mammal, canine or other species, whenadministered in a form suitable for inducing an effective immuneresponse that inhibits the in vivo activity of endogenous RANKL protein,to provide desirable benefits, such as maintaining or increasing bonemineral density or strength. Polypeptide fragments preferably include,for example, those listed by Table 1, below.

TABLE 1 From about residue 10 to about residue 275 of SEQ ID NO: 2; Fromabout residue 30 to about residue 275 of SEQ ID NO: 2; From aboutresidue 50 to about residue 275 of SEQ ID NO: 2; From about residue 150to about residue 275 of SEQ ID NO: 2; From about residue 250 to aboutresidue 275 of SEQ ID NO: 2; From about residue 255 to about residue 275of SEQ ID NO: 2; From about residue 235 to about residue 255 of SEQ IDNO: 2; From about residue 215 to about residue 235 of SEQ ID NO: 2; Fromabout residue 195 to about residue 215 of SEQ ID NO: 2; From aboutresidue 175 to about residue 195 of SEQ ID NO: 2 From about residue 155to about residue 175 of SEQ ID NO: 2; From about residue 135 to aboutresidue 155 of SEQ ID NO: 2; From about residue 95 to about residue 135of SEQ ID NO: 2; From about residue 75 to about residue 95 of SEQ ID NO:2; From about residue 55 to about residue 75 of SEQ ID NO: 2; From aboutresidue 35 to about residue 55 of SEQ ID NO: 2; From about residue 15 toabout residue 35 of SEQ ID NO: 2; From about residue 1 to about residue15 of SEQ ID NO: 2; as well as combinations of the foregoing.

The present invention is also contemplated to include recombinantproteins, e.g., heterologous fusion proteins comprising, e.g., fragmentsof polypeptide of SEQ ID NO:2, e.g., including the above-enumeratedpolypeptide fragments. A heterologous fusion protein is a fusion ofproteins or segments which are naturally not normally fused in the samemanner. A similar concept applies to heterologous nucleic acidsequences, e.g., nucleic acid molecule(s) encoding SEQ ID NO:2, e.g.,including nucleic acid molecules encoding the above-enumeratedpolypeptide fragments. Fusion proteins will be useful as sources forcleaving, separating, and purifying portions thereof.

Fusion proteins comprising canine RANKL polypeptides and otherhomologous or heterologous proteins are prepared by recombinant and/orsynthetic peptide methods to include, e.g., a reporter polypeptide,e.g., luciferase, with a segment or domain of a protein, e.g., areceptor-binding segment, so that the presence or location of the fusedligand may be easily determined. See, e.g., Dull, et. al., U.S. Pat. No.4,859,609. Other desirable fusion partners include bacterialβ-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcoholdehydrogenase, yeast alpha mating factor, and detection or purificationtags such as a FLAG sequence of His6 sequence. See, e.g., Godowski, et.al. (1988) Science 241:812-816.

In addition, fusion proteins comprise operatively linking similarfunctional domains from other proteins using art-known methods. Forexample, target-binding or other segments may be “swapped” betweendifferent new fusion polypeptides or fragments. See, e.g., Cunningham,et al. (1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol.Chem. 263:15985-15992, as well as the fusion murine RANKL constructsdescribed by co-owned U.S. Pat. Nos. 6,242,586, and 6,525,180, thedisclosures of which are incorporated by reference herein. Theabove-provided fragments of SEQ ID NO:2 are preferably incorporated intoa fusion protein to provide an immunogenic fusion protein useful inpreparing an immunogenic composition and/or vaccine.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will also produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques. Other methods of producing fusion proteins are known,including those taught by published U.S. Patent Appl. No. 20030165996,and published WO0015807A1, the disclosures of which are incorporated byreference herein.

For example, a canine RANKL fusion protein immunogen based on thepolypeptide or polypeptide fragment of SEQ ID NO:2, includes thefollowing modifications and/or features added to SEQ ID NO: 2 or afragment thereof

-   -   (a) at least one foreign T helper lymphocyte epitope,    -   (b) at least one element that targets the canine RANKL immunogen        to an antigen presenting cell or a B-lymphocyte,    -   (c) at least one element that stimulates the immune system,    -   (d) at least one element that optimizes presentation of the        canine RANKL polypeptide to the immune system, and/or        combinations thereof.

Preferably, a substantial fraction of the original B-lymphocyte epitopesof the RANKL polypeptide are retained.

In one preferred embodiment, side groups, e.g., foreign T-cell epitopesor the first, second and third moieties noted above, are covalently ornon-covalently attached to the canine RANKL polypeptide. This isaccomplished by derivatizing one or more amino acid residues of theRANKL polypeptide without altering the primary amino acid sequence,and/or without introducing changes in the peptide bonds between theindividual polypeptide residues.

An alternative preferred embodiment provides for canine RANKL immunogenswherein the polypeptide of SEQ ID NO:2 is more extensively modified byrecombinant or peptide synthetic methods, e.g., by preparing deletion orinsertion muteins or fusion polypeptides. For example, WO 95/05849, thedisclosure of which is incorporated by reference herein in its entirety,describes a method for down-regulating self-proteins by immunising ananimal with analogs of the self-proteins. The analogs are prepared bysubstituting parts of the polypeptide of interest with a correspondingnumber of amino acid sequence(s) that comprise a foreign immunodominantT-cell epitope, while at the same time maintaining the overall tertiarystructure of the self-protein in the analog. The modification can beprovided by insertion, addition, deletion or substitution of the aminoacid residues of SEQ ID NO:2 in order to provide a RANKL immunogencomprising a foreign T-cell epitope while retaining sufficient B-cellepitopes of SEQ ID NO:2. Preferably, the overall tertiary structure ofthe canine RANKL polypeptide is maintained.

The present invention contemplates modified canine RANKL immunogensobtained by deletions of those domains of the canine RANKL sequencewhich e.g., exhibit adverse effects in vivo and/or deletion of domainsthat are normally located intracellularly, and thus could give rise toundesirable immunological reactions.

Canine RANKL immunogens retaining a substantial fraction of B-cellepitopes and the overall tertiary structure of native canine RANKLpolypeptide or an immunogenic portion thereof, can be attained in anumber of ways, even for a polypeptide modified by the methods describedsupra. One such method comprises preparing an anti-canine RANKLpolyclonal anti-serum to provide a test reagent (e.g., in a competitiveELISA) against the modified canine RANKL polypeptides. Analogs thatreact to the same extent with the antiserum as does canine RANKL can beconsidered to have the same overall tertiary structure as does nativecanine RANKL. Further, modified canine RANKL polypeptides exhibiting alimited (but still significant and specific) reactivity with such anantiserum are regarded as having maintained a substantial fraction ofthe original B-cell epitopes.

An alternative preferred method provides for monoclonal antibodiesreactive with distinct epitopes of canine RANKL that are prepared andused in a test panel. This approach has the advantage of allowing (1)epitope mapping of canine RANKL and (2) mapping of the epitopes whichare maintained in the analogs prepared.

Yet another alternative method provides for resolving the 3-dimensionalstructure of canine RANKL polypeptide, or of a biologically activetruncate thereof that is compared to the resolved three-dimensionalstructure of each of the modified polypeptides. Three-dimensionalstructural determinations can be made through X-ray diffraction studies,NMR-spectroscopy, and/or by circular dichroism studies.

Nucleic Acids and Expression Vectors

The terms “polynucleotide” or “nucleic acid”, as used herein, refer to aseries of nucleotides, e.g., deoxyribonucleic acid or ribonucleic acidbases, bound to a polymer backbone. These include, for example, genomicDNA, cDNA, RNA, mRNA, any synthetic and genetically manipulatedpolynucleotides, and both sense and antisense polynucleotides. This termalso includes single and double stranded molecules; i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids. Typical nucleotides include inosine,adenosine, guanosine, cytosine, uracil and thymidine. However, nucleicacids may also contain modified nucleotide bases, for example,thio-uracil, thio-guanine and fluor-uracil.

A polynucleotide or nucleic acid may be flanked by natural regulatorysequences or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenlation sequences, introns, 5′ and 3′ non-coding regions and thelike. The nucleic acids may also be modified by any means known in theart. Non-limiting examples of such modification include methylation,caps and substitution of one or more of the naturally occurringnucleotides with an analog. Polynucleotides may contain one or moreadditional covalently linked moieties, such as proteins, intercalators,chelators and alkylators. Furthermore the polynucleotides may also bemodified with a label capable of providing a detectable signal, eitherdirectly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin and the like.

The term “recombinant” defines a biological material (e.g., a nucleicacid or polypeptide) either by its method of production or itsstructure. For example, some recombinant nucleic acids are made by theuse of recombinant DNA techniques which involve human intervention,either in manipulation or selection. Other recombinant nucleic acids aremade by fusing two nucleotide fragments that are not naturallycontiguous to each other. Engineered vectors are encompassed, as well asnucleic acids comprising sequences derived using any syntheticoligonucleotide process.

The present invention further encompasses recombinant DNA molecules andfragments having sequences that are identical or highly homologous tothose described herein, excluding those nucleotide sequences that encodehuman, murine, and rat RANKL.

“Identity”, as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide. “Identity” and “similarity” can be readily calculated byknown methods, including, but not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo,H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO: 1, that is itmay be 100% identical, or it may include up to a certain integer numberof nucleotide alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected form the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of nucleotides in SEQ ID NO: 1 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of nucleotides in SEQ ID NO: 1, orn_(n)=x_(n)−(x_(n)*y), wherein n_(n) is the number of nucleotidealterations, x_(n) is the total number of nucleotides in SEQ ID NO: 1, yis, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., * is thesymbol for the multiplication operator, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n).

Preferred polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least about 50, 60, 70,80, 85, 90, 95, 97 or 100% identity to the polypeptide referencesequence of SEQ ID NO: 2, wherein said polypeptide sequence may beidentical to the reference sequence of SEQ ID NO: 2 or may include up toa certain integer number of amino acid alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence, and wherein said number of aminoacid alterations is determined by multiplying the total number of aminoacids in SEQ ID NO: 2 by the integer defining the percent identitydivided by 100 and then subtracting that product from said total numberof amino acids in SEQ ID NO: 2, or n_(a)=x_(a)−(x_(a)*y), wherein n_(a)is the number of amino acid alterations, x_(a) is the total number ofamino acids in SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and * is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO: 2, that is it maybe 100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given percent identity is determined bymultiplying the total number of amino acids in SEQ ID NO: 2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO: 2, or n_(a)=x_(a)−(x_(a)*y), wherein n_(a) is the number of aminoacid alterations, x_(a) is the total number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.,and * is the symbol for the multiplication operator, and wherein anynon-integer product of x_(a) and y is rounded down to the nearestinteger prior to subtracting it from x_(a).

The term “homology”, as it is used herein, embraces both identity andsimilarity.

For example, some of the variants have substantial amino acid sequencehomology with the amino acid sequence of the canine RANK ligand. In thisinvention, amino acid sequence homology or sequence identity isdetermined by optimizing residue matches and, if necessary, byintroducing gaps as required. Homologous amino acid sequences aretypically intended to include natural allelic, polymorphic andinterspecies variations in each respective sequence. Typical homologousproteins or polypeptides will have from 25-100% homology (if gaps can beintroduced) to 50-100% homology (if conservative substitutions areincluded) with the amino acid sequence of canine RANKL. Observedhomologies will typically be at least about 35%, preferably at leastabout 50%, more preferably at least about 75%, and most preferably atleast about 80% or more. See, Needleham et al., J. Mol. Biol. 48:443-453(1970); Sankoff et al. in Time Warps, String Edits, and Macromolecules:The Theory and Practice of Sequence Comparison, 1983, Addison-Wesley,Reading, Mass.; and software packages from IntelliGenetics, MountainView, Calif., and the University of Wisconsin Genetics Computer Group,Madison, Wis.

Homologous nucleic acid sequences are those which when aligned andcompared exhibit significant similarities. Standards for homology innucleic acids are either measures for homology generally used in the artby sequence comparison or based upon hybridization conditions, which aredescribed in greater detail below.

Substantial nucleotide sequence homology is observed when there isidentity in nucleotide residues in two sequences (or in theircomplementary strands) when optimally aligned to account for nucleotideinsertions or deletions, in at least about 50%, preferably in at leastabout 75%, more preferably in at least about 90%, and most preferably inat least about 95% of the aligned nucleotides. Substantial homology alsoexists when one sequence will hybridize under selective hybridizationconditions to another. Typically, selective hybridization will occurwhen there is at least about 55% homology over a stretch of at leastabout 30 nucleotides, preferably at least about 65% homology over astretch of at least about 25 nucleotides, more preferably at least about75% homology, and most preferably at least about 90% homology over about20 nucleotides. See, e.g., Kanehisa, Nucleic Acids Res. 12:203 (1984).The lengths of such homology comparisons may encompass longer stretchesand in certain embodiments may cover a sequence of at least about 17,preferably at least about 25, more preferably at least about 50, andmost preferably at least about 75 nucleotide residues.

Stringency conditions employed in hybridizations to establish homologyare dependent upon factors such as salt concentration, temperature, thepresence of organic solvents and other parameters. Stringent temperatureconditions usually include temperatures in excess of about 30° C., oftenin excess of about 37° C., typically in excess of about 45° C.,preferably in excess of about 55° C., more preferably in excess of about65° C. and most preferably in excess of about 70° C. Stringent saltconditions will ordinarily be less than about 1000 mM, usually less thanabout 500 mM, more usually less than about 400 mM, preferably less thanabout 300 mM, more preferably less than about 200 mM and most preferablyless than about 150 mM. For example, salt concentrations of 100, 50 and20 mM are used. The combination of the foregoing parameters, however, ismore important than the measure of any single parameter. See, e.g.,Wetmur et al., J. Mol. Biol. 31:349 (1968).

A further indication that two nucleic acid sequences encodingpolypeptides are substantially identical is that the polypeptide encodedby the first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions.

Nucleic acids encoding canine RANKL or fragments thereof can be preparedby standard methods. For example, DNA can be chemically synthesizedusing, e.g., the phosphoramidite solid support method of Matteucci etal. [J. Am. Chem. Soc. 103:3185 (1981)], the method of Yoo et al [J.Biol. Chem. 764:17078 (1989)], or other well known methods.

Of course, due to the degeneracy of the genetic code, many differentnucleotide sequences can encode the canine RANK ligand. The codons canbe selected for optimal expression in prokaryotic or eukaryotic systems.Such degenerate variants are, of course, also encompassed by the presentinvention.

Moreover, nucleic acids encoding the canine RANK ligand can readily bemodified by nucleotide substitutions, nucleotide deletions, nucleotideinsertions, and inversions of nucleotide stretches. Such modificationsresult in novel DNA sequences that encode antigens having immunogenic orantigenic activity in common with the wild-type ligand. These modifiedsequences can be used to produce wild type or mutant ligands, or toenhance expression in a recombinant DNA system.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors that can be used in this invention include microbialplasmids, viruses, bacteriophage, integratable DNA fragments and othervehicles that may facilitate integration of the nucleic acids into thegenome of the host. Plasmids are the most commonly used form of vector,but all other forms of vectors which serve an equivalent function andwhich are or become known in the art are suitable for use herein. See,e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 andSupplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth,Boston, Mass.

Insertion of DNA encoding the canine RANK ligand into a vector is easilyaccomplished when the termini of both the DNA and the vector comprisecompatible restriction sites. If this cannot be done, it may benecessary to modify the termini of the DNA and/or vector by digestingback single-stranded DNA overhangs generated by restriction endonucleasecleavage to produce blunt ends, or to achieve the same result by fillingin the single-stranded termini with an appropriate DNA polymerase.Alternatively, desired sites may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated through theuse of the polymerase chain reaction (PCR). See, e.g., Saiki et al.,Science 239:487 (1988). The cleaved vector and the DNA fragments mayalso be modified, if required, by homopolymeric tailing.

Recombinant expression vectors used in this invention are typicallyself-replicating DNA or RNA constructs comprising nucleic acids encodingone of the canine RANK ligands, usually operably linked to suitablegenetic control elements that are capable of regulating expression ofthe nucleic acids in compatible host cells. Genetic control elements mayinclude a prokaryotic promoter system or a eukaryotic promoterexpression control system, and typically include a transcriptionalpromoter, an optional operator to control the onset of transcription,transcription enhancers to elevate the level of mRNA expression, asequence that encodes a suitable ribosome binding site, and sequencesthat terminate transcription and translation. Expression vectors mayalso contain an origin of replication that allows the vector toreplicate independently of the host cell.

Expression of nucleic acids encoding the canine RANK ligand of thisinvention can be carried out by conventional methods in eitherprokaryotic or eukaryotic cells. The term “host cell” means any cellthat can express a foreign gene, DNA or RNA sequence to produce adesired substance, such as an RNA or protein. Suitable host cells forexpressing nucleic acids encoding canine RANKL include prokaryotes andhigher eukaryotes. Prokaryotes include both gram negative and positiveorganisms, e.g., E. coli and B. subtilis. Higher eukaryotes includeestablished tissue culture cell lines from animal cells, both ofnon-mammalian origin, e.g., insect cells, and birds, and of mammalianorigin, e.g., human, primates, and rodents.

Prokaryotic expression control sequences typically used includepromoters, including those derived from the β-lactamase and lactosepromoter systems [Chang et al., Nature, 198:1056 (1977)], the tryptophan(trp) promoter system [Goeddel et al., Nucleic Acids Res. 8:4057(1980)], the lambda P_(L) promoter system [Shimatake et al., Nature,292:128 (1981)] and the tac promoter [De Boer et al., Proc. Natl. Acad.Sci. USA 292:128 (1983)]. Numerous expression vectors containing suchcontrol sequences are known in the art and commercially available.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express canineRANKL include, but are not limited to, those containing the lac promoter(pUC-series); trp promoter (pBR322-trp); Ipp promoter (the pIN-series);lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac(pDR540). See, Brosius et al., “Expression Vectors Employing Lambda-,trp-, lac-, and Ipp-derived Promoters”, in Rodriguez and Denhardt (eds.)Vectors: A Survey of Molecular Cloning Vectors and Their Uses, 1988,Buttersworth, Boston, pp. 205-236.

Higher eukaryotic tissue culture cells are preferred hosts for therecombinant production of canine RANKL. Although any higher eukaryotictissue culture cell line might be used, including insect baculovirusexpression systems, mammalian cells are preferred. Transformation ortransfection and propagation of such cells have become a routineprocedure. Examples of useful cell lines include HeLa cells, Chinesehamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insectcell lines, bird cell lines, and monkey (COS) cell lines.

Expression vectors for such cell lines usually include an origin ofreplication, a promoter, a translation initiation site, RNA splice sites(if genomic DNA is used), a polyadenylation site, and a transcriptiontermination site. These vectors also usually contain a selection gene oramplification gene. Suitable expression vectors may be plasmids,viruses, or retroviruses carrying promoters derived, e.g., from suchsources as adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pCR®3.1, pcDNA1, pCD [Okayama et al., Mol. Cell Biol. 5:1136(1985)], pMC1neo Poly-A [Thomas et al., Cell 51:503 (1987)], pUC19,pREP8, pSVSPORT and derivatives thereof, and baculovirus vectors, suchas pAC 373 or pAC 610.

Protein Purification

The proteins, polypeptides and antigenic fragments of this invention canbe purified by standard methods including, but not limited to, salt oralcohol precipitation, preparative disc-gel electrophoresis, isoelectricfocusing, high pressure liquid chromatography (HPLC), reversed-phaseHPLC, gel filtration, cation and anion exchange, partitionchromatography and countercurrent distribution. Such purificationmethods are well known in the art and are disclosed, e.g., in Guide toProtein Purification, Methods in Enzymology, Vol. 182, M. Deutscher,Ed., 1990, Academic Press, New York, N.Y.

Purification steps can be followed by carrying out assays for ligandbinding activity as described below. Particularly where a ligand isbeing isolated from a cellular or tissue source, it is preferable toinclude one or more inhibitors of proteolytic enzymes is the assaysystem, such as phenylmethanesulfonyl fluoride (PMSF).

Screening Systems and Methods

The present invention allows for the discovery of selective antagonistsof the canine RANK ligand that may be useful in the treatment andmanagement of a variety of diseases including inflammation, bonedisease, osteoarthritis, rheumatoid arthritis, osteoporosis and pain.Thus, a ligand of the present invention can be employed in screeningsystems to identify antagonists of RANKL. Essentially, these systemsprovide methods for bringing together a RANK receptor, an appropriateligand, including canine RANK ligand itself, and a sample to be testedfor the presence of a canine RANKL antagonist.

Two basic types of screening systems can be used, a labeled-ligandbinding assay and a “functional” assay. A labeled ligand for use in thebinding assay can be obtained by labeling canine RANKL with a measurablegroup, as described below in connection with the labeling of antibodies.Various labeled forms of canine RANKL can be generated using standardtechniques. Alternatively, the RANK receptor can be labeled.

Typically, a given amount of the RANK receptor is contacted withincreasing amounts of a labeled ligand, such as labeled canine RANKLitself, and the amount of the bound labeled ligand is measured afterremoving unbound labeled ligand by washing. As the amount of the labeledligand is increased, a point is eventually reached at which all RANKreceptor binding sites are occupied or saturated. Specific receptorbinding of the labeled ligand is abolished by a large excess ofunlabeled ligand.

Preferably, an assay system is used in which non-specific binding of thelabeled ligand to the RANK receptor is minimal. Non-specific binding istypically less than 50%, preferably less than 15%, and more preferablyless than 10% of the total binding of the labeled ligand.

In principle, a binding assay of the present invention could be carriedout using a soluble RANK receptor and the resulting receptor-labeledligand complex could be precipitated, e.g., using an antibody againstthe ligand. The precipitate could then be washed and the amount of thebound labeled ligand could be measured.

Preferably, however, a RANK receptor is incorporated into the membraneof a cell. A membrane fraction can then be isolated from the cell andused as a source of the receptor for assay.

The binding assays of this invention can be used to identify antagonistsof canine RANKL because they will interfere with the binding of theligand to the RANK receptor.

In the basic binding assay, a method for identifying a canine RANKLantagonist comprises:

(a) contacting a RANK receptor or a subsequence thereof, in the presenceof a known amount of canine RANKL with a sample to be tested for thepresence of a canine RANKL antagonist; and

(b) measuring the amount of canine RANKL bound to the receptor;

whereby a canine RANKL antagonist in the sample is identified bymeasuring substantially reduced binding of the RANKL to the RANKreceptor, compared to what would be measured in the absence of suchantagonist. As stated previously, either the RANKL or RANK may belabeled.

Determination of whether a particular molecule inhibiting binding of thecanine RANK ligand to the RANK receptor is an antagonist or an agonistis then made in a second, functional assay. The functionality ofmolecules identified in the binding assay can be determined in cellularand animal models.

In cellular models, parameters for intracellular activities mediated byRANKL can be monitored. Such parameters include, but are not limited to,altered intracellular cAMP or Ca²⁺ concentrations. Methods using animalsor animal tissues for such activities can also be employed.

In the basic functional assay, a method for identifying an antagonist ofa canine RANK ligand comprises:

(a) contacting cells expressing the canine RANKL polypeptide in thepresence of a known amount of RANK or surrogate thereof with a sample tobe tested for the presence of a canine RANK ligand antagonist; and

(b) measuring at least one cellular function modulated by the binding ofRANK to the polypeptide;

whereby a canine RANK ligand antagonist in the sample is identified bymeasuring substantially reduced effects on said cellular functioncompared to what would be measured in the absence of such antagonist.

Mammalian RANK Ligands from Other Species

The present invention provides methods for cloning RANK ligands fromother species. Briefly, Southern and Northern blot analysis can beperformed to identify cells from other species expressing genes encodingthe RANK ligand. Complementary DNA (cDNA) libraries can be prepared bystandard methods from mRNA isolated from such cells, and degenerateprobes or PCR primers based on the nucleic acid and amino acid sequencesprovided herein can be used to identify clones encoding a RANK ligand.

Alternatively, expression cloning methodology can be used to identifyparticular clones encoding a RANK ligand. An antibody preparation whichexhibits cross-reactivity with RANK ligands from a number of mammalianspecies may be useful in monitoring expression cloning.

However identified, clones encoding RANK ligand from various species canbe isolated and sequenced, and the coding regions can be excised andinserted into an appropriate vector.

Localization of mRNA Encoding the Polypeptide of SEQ ID NO: 2

The present invention also provides compositions and methods forlocalization of messenger RNA coding for the polypeptide defined by theamino acid sequence of SEQ ID NO: 2.

Specifically, cell line blots containing approximately 2 μg of poly(A)⁺RNA per lane are purchased from Clontech (Palo Alto, Calif.). Probes areradiolabeled with [α³²P] dATP, e.g., using the Amersham Rediprime randomprimer labeling kit (RPN1633). Prehybridization and hybridizations areperformed at 65° C. in 0.5 M Na₂HPO₄, 7% SDS and 0.5 mM EDTA (pH 8.0).High stringency washes are conducted, e.g., at 65° C. with an initialwash in 6×SSC, 0.1% SDS for 15 min followed by two subsequent washes in0.2×SSC, 0.1% SDS for 15 min. The mixture is then exposed at −70° C. toX-Ray film (Kodak) in the presence of intensifying screens. Moredetailed studies by cDNA library Southerns are performed with selectedclones of nucleic acids having the nucleotide sequence defined by SEQ IDNO: 1 to examine their expression in other cell subsets.

Two prediction algorithms that take advantage of the patterns ofconservation and variation in multiply aligned sequences, (Rost andSander (1994) Proteins 19:55-72) and DSC (King and Stemberg (1996)Protein Sci. 5:2298-2310), are used.

Alternatively, two appropriate primers are selected and RT-PCR is usedon an appropriate mRNA sample selected for the presence of message toproduce a cDNA, e.g., a sample which expresses the gene.

Full length clones may be isolated by hybridization of cDNA librariesfrom appropriate tissues pre-selected by PCR signal.

Message for nucleic acids encoding a polypeptide having the amino acidsequence of SEQ ID NO: 2 are assayed by appropriate technology, e.g.,PCR, immunoassay, hybridization or otherwise. Tissue and organ cDNApreparations are available, e.g., from Clontech, Mountain View, Calif.

Southern Analysis on cDNA libraries is performed as follows: DNA (5 μg)from a primary amplified cDNA library is digested with appropriaterestriction enzymes to release the inserts, run on a 1% agarose gel andtransferred to a nylon membrane (Schleicher and Schuell, Keene, N.H.).

Immunogenic Compositions. Vaccines, and Antibody Production

An “immunogenic composition” is a substance or a combination ofsubstances, covalently or noncovalently combined, effective to elicit animmune response in an organism and/or from isolated immune system cells.An immune response is the reaction of the body to foreign substances,without implying a physiologic or pathologic consequence of such areaction, i.e., without necessarily conferring protective immunity onthe organism. An immune response may include one or more of thefollowing: a cell mediated immune response, which involves theproduction of lymphocytes by the thymus in response to exposure to anantigen; and/or a humoral immune response, which involves production ofplasma lymphocytes in response to antigen exposure with subsequentantibody production. Immunogenic compositions are useful as antigens toelicit the production of antibodies.

Antigenic (i.e., immunogenic) fragments of the canine RANK ligand ofthis invention, which may or may not have RANK receptor bindingactivity, may be produced as immunogens. Regardless of whether they bindRANK, such fragments, like the complete ligands, are useful as antigensfor preparing antibodies, by standard methods, that can prevent bindingto the receptors. Because it is well known in the art that epitopesgenerally contain at least about five, preferably at least about 8,amino acid residues [Ohno et al., Proc. Natl. Acad. Sci. USA 82:2945(1985)], fragments used for the production of antibodies will generallybe at least that size. Preferably, they will contain even more residues.Whether a given fragment is immunogenic can readily be determined byroutine experimentation. Simply by way of example, preferred fragmentsof the canine RANKL polypeptide fragments include those listed by Table1, supra.

The artisan will also appreciate that selection of desirable immunogenicfragments/epitopes of a polypeptide depends upon the tertiary structureof the polypeptide. Preferred fragments for eliciting binding antibodiesare those peptide structures external to the folded polypeptide, e.g.peptide loops that comprise the outside or solvent-accessable portionsof the folded structure and that are accessable to the immune system orimmune cells. The polypeptide of SEQ ID NO: 2 primarily corresponds tothe extracellular portion of the endogenous, cell-membrane bound canineRANKL. Based on murine RANKL tertiary structure and homologies withinthe TNF superfamily of proteins, Lam et al. [J Clin Invest, 108(7):971-979 (2001), incorporated herein by reference] have reported,that the externally presented loops that are unique to murine RANKL areclustered in the solvent-accessible AA″, CD, DE, and EF loopsillustrated by FIG. 2 of the Lam et al. article (Id.). It iscontemplated that the analogous regions of the canine RANKL of SEQ IDNO: 2 are preferred, but not exclusive, domains for antibody recognitionand selective binding of the inventive canine RANKL polypeptide. Thus,canine RANKL polypeptides that are preferred as immunogens and/or ascomponents of suitable fusion proteins and/or other vaccine preparationsinclude, for example, a canine RANKL polypeptide fragment correspondingto the entire murine RANKL loop region, from about residue 110 to aboutresidue 140 of SEQ ID NO:2, and well as the particular canine RANKLpolypeptides listed by Table 2, below.

TABLE 2 From about residue 125 to about residue 160 of SEQ ID NO: 2;From about residue 119 to about residue 153 of SEQ ID NO: 2; From aboutresidue 175 to about residue 200 of SEQ ID NO: 2; From about residue 183to about residue 192 of SEQ ID NO: 2; From about residue 200 to aboutresidue 225 of SEQ ID NO: 2; From about residue 204 to about residue 211of SEQ ID NO: 2; From about residue 195 to about residue 215 of SEQ IDNO: 2; From about residue 221 to about residue 227 of SEQ ID NO: 2; aswell as combinations of the foregoing.

Preferably, canine RANKL polypeptide and/or fragments, e.g., of Tables 1and/or 2, are cross-linked to a carrier molecule to enhance theirimmunogenicity via a “carrier effect.” Conjugation polypeptide and/orpolypeptide fragments to an immunogenic carrier molecule renders themmore immunogenic through what is commonly known as the “carrier effect”.This is particularly preferred for extracellular polypeptides to whichthe treated mammal is typically immunologically tolerant.

Suitable carrier molecules include, e.g., proteins and natural orsynthetic polymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides, etc. Protein carrier molecules are especiallypreferred, including, but not limited to, keyhole limpet hemocyanin andmammalian serum proteins, such as human or bovine gamma-globulin, human,bovine or rabbit serum albumin, or methylated or other derivatives ofsuch proteins. Other protein carriers will be apparent to those skilledin the art. Preferably, but not necessarily, the protein carrier will beforeign to the host animal in which antibodies against the fragments areto be elicited.

Covalent coupling to the carrier molecule can be achieved using methodswell known in the art, the exact choice of which will be dictated by thenature of the carrier molecule used. When the immunogenic carriermolecule is a protein, the fragments of the present invention can becoupled, e.g., using water-soluble carbodiimides, such asdicyclohexylcarbodiimide or glutaraldehyde. Coupling agents such asthese can also be used to cross-link the fragments to themselves withoutthe use of a separate carrier molecule. Such cross-linking intoaggregates can also increase immunogenicity.

Immunogenicity can also be increased by the use of adjuvants, alone orin combination with coupling or aggregation. Suitable adjuvants for thevaccination of animals include, but are not limited to, Adjuvant 65(containing peanut oil, mannide monooleate and aluminum monostearate);Freund's complete or incomplete adjuvant; mineral gels, such as aluminumhydroxide, aluminum phosphate and alum; surfactants, such ashexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis(2-hydroxymethyl) propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions, such aspyran, dextran sulfate, poly IC, polyacrylic acid and carbopol;peptides, such as muramyl dipeptide, dimethylglycine and tuftsin; andoil emulsions. The polypeptides could also be administered followingincorporation into liposomes or other microcarriers. Informationconcerning adjuvants and various aspects of immunoassays are disclosed,e.g., in the series by P. Tijssen, Practice and Theory of EnzymeImmunoassays, 3rd Edition, 1987, Elsevier, New York.

A vaccine (or vaccine composition) can be an antigen and/or acomposition, including an immuogenic composition described above, and/ora formulation that when administered to a subject animal engenders animmunogenic challenge in a controlled manner in that subject animal.Vaccines of the present invention, for example, can comprise an antigenor an expressible nucleic acid (e.g., in a viral vector or naked DNAvector) that encodes an antigen. In a particular embodiment, the antigenis the canine RANKL polypeptide or an immunogenic fragment thereof. Allof the forms of the canine RANKL polypeptide and immunogenic fragmentsthereof taught by the present invention may be part of such a vaccine.In a particular embodiment, administering a vaccine of the presentinvention to an animal subject serves to negate, at least in part, thebiological action (activity) of the native RANKL of the subject animal.Vaccines of the present invention generally, but by no means always,comprise an adjuvant as exemplified above. Examples of methods ofadministering vaccines contemplated by the present invention includesubcutaneous, parenteral, intraperitoneal, scarification, intravenous,intramuscular injection and infusion. The formulation, use andadministration of vaccines are well known in the art and have beenreviewed in published PCT application WO00/158071 and U.S. Pat. No.6,645,500 B1, the contents of which are hereby incorporated by referencein their entireties.

The present invention also includes polyclonal and monoclonal (mAb)antibodies that bind to canine RANKL, and preferably antibodies thatbind specifically to canine RANKL. As used herein, the term “antibody”refers to an immunoglobulin and/or fragments thereof. A naturallyoccurring immunoglbulin consists of one or more polypeptidessubstantially encoded by immunoglobulin genes. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. An antibody or antibodiesaccording to the present invention also encompass antibody fragments,i.e., antigen-binding fragments, for example, Fv, Fab, and F(ab′)₂,engineered single-chain binding proteins, e.g., Huston et al., Proc.Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science,242,423426 (1988), incorporated herein by reference herein), as well asbifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J.Immunol. 17, 105 (1987)). See, generally, Hood et al., Immunology,Benjamin, N.Y., 2nd ed. (1984), Harlow and Lane, Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapillerand Hood, Nature, 323, 15-16 (1986), all of which are incorporated byreference herein.

For example, serum produced from animals immunized using standardmethods can be used directly, or the IgG fraction can be separated fromthe serum using standard methods, such as plasmaphoresis or adsorptionchromatography with IgG-specific adsorbents, such as immobilized ProteinA. Alternatively, monoclonal antibodies can be prepared, and optionally,antigen binding fragments or recombinant binding proteins derived fromsuch mAbs. Such MAbs or fragments thereof can be humanized by art-knownmethods if so desired.

Hybridomas producing mAbs that selectively bind canine RANKL of thepresent invention, or antigenic fragments of canine RANKL, are producedby well-known techniques. Usually, the process involves the fusion of animmortalizing cell line with a B-lymphocyte that produces the desiredantibody. Alternatively, non-fusion techniques for generating immortalantibody-producing cell lines can be used, e.g., virally-inducedtransformation [Casali et al., Science 234:476 (1986)]. Immortalizingcell lines are usually transformed mammalian cells, particularly myelomacells of rodent, bovine, and human origin. Most frequently, rat or mousemyeloma cell lines are employed as a matter of convenience andavailability.

Techniques for obtaining antibody-producing lymphocytes from mammalsinjected with antigens are well known. Generally, peripheral bloodlymphocytes (PBLs) are used if cells of human origin are employed, orspleen or lymph node cells are used from non-human mammalian sources. Ahost animal is injected with repeated dosages of the purified antigen(human cells are sensitized in vitro), and the animal is permitted togenerate the desired antibody-producing cells before they are harvestedfor fusion with the immortalizing cell line. Techniques for fusion arealso well known in the art, and, in general, involve mixing the cellswith a fusing agent, such as polyethylene glycol.

Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. Those secreting thedesired antibody are selected using standard immunoassays, such asWestern blotting, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay) or the like. Antibodies are recovered from the mediumusing standard protein purification techniques [Tijssen, Practice andTheory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)].

Many references are available to provide guidance in applying the abovetechniques [Kohler et al., Hybridoma Techniques (Cold Spring HarborLaboratory, New York, 1980); Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal AntibodyTechnology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal HybridomaAntibodies: Techniques and Applications (CRC Press, Boca Raton, Fla.,1982)]. Monoclonal antibodies can also be produced using well-knownphage library systems. See, e.g., Huse, et al., Science 246:1275 (1989);Ward, et al., Nature, 341:544 (1989).

Antibodies thus produced, whether polyclonal or monoclonal, can be used,e.g., in an immobilized form bound to a solid support by well knownmethods to purify the ligands by immunoaffinity chromatography.

Antibodies against the antigenic fragments can also be used, unlabeledor labeled by standard methods, as the basis for immunoassays of thecanine RANK ligand. The particular label used will depend upon the typeof immunoassay. Examples of labels that can be used include, but are notlimited to, radiolabels, such as ³²P, ¹²⁵I, ³H and ¹⁴C; fluorescentlabels, such as fluorescein and its derivatives, rhodamine and itsderivatives, dansyl and umbelliferone; chemiluminescers, such asluciferia and 2,3-dihydrophthalazinediones; and enzymes, such ashorseradish peroxidase, alkaline phosphatase, lysozyme andglucose-6-phosphate dehydrogenase.

The antibodies can be tagged with such labels by known methods. Forexample, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bisdiazotized benzadine and the like may be usedto tag the antibodies with fluorescent, chemiluminescent or enzymelabels. The general methods involved are well known in the art and aredescribed, e.g., in Immunoassay: A Practical Guide, 1987, Chan (Ed.),Academic Press, Inc., Orlando, Fla. Such immunoassays could be carriedout, for example, on fractions obtained during purification of thereceptors.

The antibodies of the present invention can also be used to identifyparticular cDNA clones expressing canine RANKL in expression cloningsystems.

Neutralizing antibodies specific for the ligand-binding site of areceptor can also be used as antagonists (inhibitors) to block RANKLbinding. Such neutralizing antibodies can readily be identified throughroutine experimentation.

Antagonism of RANKL activity can be accomplished using complete antibodymolecules, or well-known antigen binding fragments such as Fab, Fc,F(ab)₂, and Fv fragments. Definitions of such fragments can be found,e.g., in Klein, Immunology (John Wiley, New York, 1982); Parham, Chapter14, in Weir, ed. Immunochemistry, 4th Ed. (Blackwell ScientificPublishers, Oxford, 1986). The use and generation of antibody fragmentshas also been described, e.g.: Fab fragments [Tijssen, Practice andTheory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)], Fv fragments[Hochman et al., Biochemistry 12:1130 (1973); Sharon et al.,Biochemistry 15:1591 (1976); Ehrlich et al., U.S. Pat. No. 4,355,023]and antibody half molecules (Auditore-Hargreaves, U.S. Pat. No.4,470,925). Methods for making recombinant Fv fragments based on knownantibody heavy and light chain variable region sequences have furtherbeen described, e.g., by Moore et al. (U.S. Pat. No. 4,642,334) and byPlückthun [Bio/Technology 9:545 (1991)]. Alternatively, they can bechemically synthesized by standard methods.

The present invention also encompasses anti-idiotypic antibodies, bothpolyclonal and monoclonal, which are produced using the above-describedantibodies as antigens. These antibodies are useful because they maymimic the structures of the ligands.

Pharmaceutical Compositions

The canine RANK ligand antagonists of this invention can be usedtherapeutically to block the activity of RANK ligand, and thereby totreat any medical condition caused or mediated by the RANK/RANKL system.The dosage regimen involved in a therapeutic application will bedetermined by the attending veterinarian, considering various factorswhich may modify the action of the therapeutic substance, e.g., thecondition, body weight, sex and diet of the patient, time ofadministration and other clinical factors.

Typical protocols for the therapeutic administration of such substancesare well known in the art. Administration of the pharmaceuticalcompositions of the present invention is typically by parenteral,intraperitoneal, intravenous, subcutaneous, intramuscular injection,infusion or any other acceptable systemic method. Often, treatmentdosages are titrated upward from a low level to optimize safety andefficacy.

Dosages will be adjusted to account for the smaller molecular sizes andpossibly decreased half-lives (clearance times) followingadministration. It will be appreciated by those skilled in the art,however, that the canine RANKL antagonists of the present inventionencompass neutralizing antibodies or binding fragments thereof inaddition to other types of inhibitors, including small organic moleculesand inhibitory ligand analogs, which can be identified using the methodsof the present invention.

An “effective amount” of a composition of the present invention is anamount that will ameliorate one or more of the well-known parametersthat characterize medical conditions caused or mediated by RANKL.

Although the compositions of this invention could be administered insimple solution, they are more typically used in combination with othermaterials such as carriers, preferably pharmaceutical carriers. Usefulpharmaceutical carriers can be any compatible, non-toxic substancessuitable for delivering the compositions of the present invention to apatient. Sterile water, alcohol, fats, waxes, and inert solids may beincluded in a carrier. Pharmaceutically acceptable adjuvants (bufferingagents, dispersing agents) may also be incorporated into thepharmaceutical composition. Generally, compositions useful forparenteral administration of such drugs are well known; e.g.,Remington's Pharmaceutical Science, 17th Ed. (Mack Publishing Company,Easton, Pa., 1990). Alternatively, compositions of the present inventionmay be introduced into a patient's body by implantable drug deliverysystems [Urquhart et al., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984)].

Therapeutic formulations may be administered in many conventional dosageformulations. Formulations typically comprise at least one activeingredient, together with one or more pharmaceutically acceptablecarriers. Formulations may include those suitable for oral, rectal,nasal or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any method well known in the art of pharmacy. See,e.g., Gilman et al. (eds.) (1990), The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, supra, Easton, Pa.; Avis et al. (eds.) (1993) PharmaceuticalDosage Forms: Parenteral Medications Dekker, New York; Lieberman et al.,(eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; andLieberman et al., (eds.) (1990), Pharmaceutical Dosage Forms: DisperseSystems Dekker, N.Y.

Anti-Sense Molecules

The present invention also encompasses anti-sense oligonucleotidescapable of specifically hybridizing to mRNA encoding a canine RANKligand having an amino acid sequence defined by SEQ ID NO: 2 or asubsequence thereof so as to prevent translation of the mRNA.Additionally, this invention contemplates anti-sense oligonucleotidescapable of specifically hybridizing to the genomic DNA molecule encodinga canine RANKL having an amino acid sequence defined by SEQ ID NO: 2 ora subsequence thereof.

This invention further provides pharmaceutical compositions comprising(a) an amount of an oligonucleotide effective to reduce activity ofcanine RANKL by passing through a cell membrane and binding specificallywith mRNA encoding canine RANKL in the cell so as to prevent itstranslation and (b) a pharmaceutically acceptable carrier capable ofpassing through a cell membrane. In an embodiment, the oligonucleotideis coupled to a substance that inactivates mRNA. In another embodiment,the substance that inactivates mRNA is a ribozyme.

The present invention may be better understood by reference to thefollowing non-limiting examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate embodiments of the present invention and should in no way beconstrued as limiting the broad scope of the present invention.

EXAMPLES

Unless otherwise indicated, percentages given below for solids in solidmixtures, liquids in liquids, and solids in liquids are on a wt/wt,vol/vol and wt/vol basis, respectively. Sterile conditions weregenerally maintained during cell culture.

Materials and General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.,(1989) Molecular Cloning: A Laboratory Manual (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al., (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene and Wiley, New York; Innis, etal. (eds.)(1990) PCR Protocols: A Guide to Methods and Applications,Academic Press, N.Y.

Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification” in Methods in Enzymology vol. 182, and other volumes inthis series; and manufacturer's literature on use of proteinpurification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad,Richmond, Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See, e.g., Hochuli(1989) Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;and Crowe, et al. (1992) OIAexpress: The High Level Expression & ProteinPurification System QIAGEN, Inc., Chatsworth, Calif.

Cell culture techniques are described in Doyle, et al. (eds.) (1994)Cell and Tissue Culture: Laboratory Procedures, John Wiley and Sons, NY.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y. Fluorescent labelingof appropriate reagents can be performed by standard methods.

Example 1 Cloning of Canine RANKL

Canine RANKL was cloned using a series of nested PCR strategies.

Nested PCR involves two sequential PCR reactions. Each PCR reactiongenerally contained 0.02 μg/μl of nucleic acid template, 1×PCR buffer,0.8 mM dNTP's, 1.1 mM Mg(OAC)₂, 0.16 units/μl of rTth polymerase(recombinant thermostable Taq polymerase), 2 OD/ml of vector primer, and0.2 OD/ml of gene specific primer. In the initial reaction, 30 cycles ofPCR were performed using a vector specific primer and a gene specificprimer (same or cross species primer). First, the reaction mixture washeated to 94° C. for 1 minute. The reaction mixture was then cycled 30times; each cycle the reaction mixture being heated to 94° C. for 1minute, then cooled to 65° C. for 5 minutes, followed by heating to 72°C. for 10 minutes. After completing 30 cycles, the reaction mixtureremained at 72° C. for 10 minutes. Subsequently, a small aliquot of thePCR product of the first reaction served as the template for a secondPCR reaction. The second PCR reaction used gene specific primers (sameor cross species) that hybridized to sequences internal to or nestedbetween the first set of primers. This is called double nesting.However, in some cases, the first reaction gene specific primer was usedin the second set of reactions with a different gene specific primer(same or cross species) for a single nesting reaction. The second PCRreaction was cycled according to the same protocol as the firstreaction.

A canine splenocyte activated cDNA library of primary DNA was preparedfrom canine splenocytes, with Concanavalin A (ConA) activiation,according to the method of Bolin et al., (1997), The Journal ofNeuroscience, 17(14):5493-5502, incorporated by reference here in itsentirety. Briefly, splenocytes were obtained from fresh canine spleen bystandard methods. RNA was isolated by standard techniques from separatedcanine splenocytes that were stimulated for 1, 2, 6, 12, and 24 hr withConcavalin A (Sigma, St. Louis, Mo.), interferon γ (γ-IFN) (200 U/ml)(Schering Plough, Kenilworth, N.J.), and anti-interleukin-10 (IL-10)antibody (10 μg/ml) (DNAX) and then pooled. Poly(A1) RNA was selectedusing oligotex beads (Qiagen, Chatsworth, Calif.). A cDNA library wasconstructed using this mRNA.

Nested PCR was performed using the GeneAmp XL PCR kit (Perkin Elmer,Branchburg, N.J.) on Canine Splenocyte activated cDNA library primaryDNA. The library may either be purchased or constructed according tomethods known in the art.

The first round PCR reaction mix contained the elements described above,with the vector specific primer being the T7 primer (SEQ ID NO: 3) andthe gene specific primer being the Rank Ligand_Human/AS2 primer (SEQ IDNO: 4). The first round PCR reaction mixture was then cycled accordingto the procedure described above.

Two μl of the first round PCR product was used as a template for asecond PCR reaction. The gene specific primers for the second reactionwere the Rank Ligand_Human/S6 primer (SEQ ID NO: 5) and the RankLigand_Human/AS4 primer (SEQ ID NO: 6). The second reaction, containingmaterials as described above, was cycled as described above.

The above two rounds of PCR generated a 1.2 kb fragment. The fragmentwas extremely faint when subjected to agarose gel analysis. The 1.2 kbfragment was then cloned into the PCRII vector (Invitrogen, Carlsbad,Calif.). The screening of transformants did not yield a clone containingthe 1.2 kb insert.

One μl of the above ligation mix (the 1.2 kb insert ligated to the PCRIIvector) was then used as template for another PCR reaction. The primersused in this reaction were the Rank Ligand_Human/S3 primer (SEQ ID NO:7) and the Rank Ligand_Human/AS4 primer (SEQ ID NO: 6). The reactionmixture was cycled 30 times according to the above described procedure.A 0.3 kb fragment was generated by this PCR reaction. The 0.3 kbfragment was then isolated by agarose gel electrophoresis and clonedinto the PCRII vector. The sequence was confirmed by analysis to be aninternal coding region of the canine RANK ligand. As is known in theart, T7 reads sense in both clones. These clones were called 01-7469A1and 01-7469A2.

The above internal coding region of the canine RANK ligand was used todesign canine specific PCR primers for subsequent nested PCR reactions.The goal was to isolate both upstream 5′ and downstream 3′ codingregions of the canine RANK ligand gene.

A 0.4 kb fragment of the 5′ upstream coding region was generated usingnested PCR. The first round reaction mixture included Canine Splenocyteactivated cDNA library primary DNA as a template, and the T7 primer (SEQID NO: 3) and the Rank Ligand_Dog/AS1 primer (SEQ ID NO: 8). The secondround reaction mixture included 2 μl of the first round reaction productas a template with Rank Ligand_Human/S6 primer (SEQ ID NO: 5) and RankLigand_Dog/AS2 primer (SEQ ID NO: 9). Both rounds of PCR were cycledaccording to the above protocol. The resulting sequence was confirmed bysequence analysis to be canine RANK ligand, excluding the 21 bpcontributed by the Human primer Rank Ligand_Human/S6. This clone wascalled 01-7557B10.

A 1.3 kb fragment of the 3′ coding region with UTR was generated usingnested PCR. The first round reaction mixture included Canine Splenocyteactivated cDNA library primary DNA as a template, and the RankLigand_Dog/S1 primer (SEQ ID NO: 10) and the vector specific primer Sp6(SEQ ID NO: 11). The second round reaction mixture included 2 μl offirst round reaction product as a template with Rank Ligand-Dog/S2primer (SEQ ID NO: 12) and vector specific primer pSPORT1 (SEQ ID NO:13). The resulting PCR product was confirmed by sequence analysis to becanine RANK ligand. This clone was called 01-7557A5.

The 1.3 kb sequence of 3′ coding region with UTR allowed for two genespecific canine RANK ligand primers, Rank Ligand-Dog/AS4 (SEQ ID NO: 14)and Rank Ligand_Dog/AS3 (SEQ ID NO: 15), to be designed in the 3′ UTRwhich made it possible to construct a nearly intact canine RANK ligandgene that could be used for protein expression.

One final nested PCR strategy was performed. The first round reactionmixture included T7 primer (SEQ ID NO: 3) and Rank Ligand_Dog/AS3 primer(SEQ ID NO: 15). The second round reaction mixture included 2 μl of thefirst round reaction product as a template with Rank Ligand_Human/S6primer (SEQ ID NO: 5) and Rank Ligand_Dog/AS4 primer (SEQ ID NO: 14).The resulting product was a 0.989 kb fragment. The 0.989 kb fragment wascloned into the PCRII vector and confirmed by sequence analysis to becanine RANK ligand, excluding the 21 bp contributed by the Human primerRank Ligand_Human/S6 (SEQ ID NO: 5). This clone was called 02-8136A5.

Example 2 Expression and Purification of Canine RANKL

Standard molecular biology techniques are used to make a chimeric DNAconstruct encoding, sequentially, residues 1-15 of the preprotrypsinsignal peptide, the FLAG™ sequence DYKDDDD (SEQ ID NO: 16),KL (encodinga HindIII site used in construction), residues VA, residues 155-319 ofthe ectodomain from canine RANKL, residues PRPPTPGNL (SEQ ID NO:17),encoding a proteolytic cleavage site), and residues 99-330 from theconstant region of human IgG gamma 1. This chimeric coding region isinserted into a modified pQB1-AdCMV5-GFP adenovirus transfer vector(Quantum Biotechnologies, Montreal, Canada) and used to make recombinantadenovirus, as previously described by Hoek et al., “Down-regulation ofthe macrophage lineage through interaction with OX2”, Science, vol. 290,pp. 1768-1771 (Dec. 1, 2000). Control adenovirus encodes the samechimeric construct minus the canine RANKL ectodomain. Recombinant Igfusion proteins are prepared using methods previously described inOppmann et al., “Novel p19 protein engages IL-12p40 to form a cytokine,IL-23, with biological activities similar as well as distinct fromIL-12”, Immunity, vol. 13, pp. 715-25 (November 2000).

It should be noted that other expression configurations can also beengineered and would be expected to result in a functional protein. Forexample, the Ig domain could be placed between the FLAG™ sequence andthe RANKL sequence. Purification could be via the identical method.

Example 3 Isolation of Homologous RANKL Genes

The canine RANKL cDNA can be used as a hybridization probe to screen alibrary from a desired source, e.g., a primate cell cDNA library. Manydifferent species can be screened both for stringency necessary for easyhybridization, and for presence using a probe. Appropriate hybridizationconditions can be used to select for clones exhibiting specificity ofcross hybridization.

Screening by hybridization or PCR using degenerate probes based upon thepeptide sequences can also allow isolation of appropriate clones.Alternatively, use of appropriate primers for PCR screening can yieldenrichment of appropriate nucleic acid clones.

Similar methods are applicable to isolate either species, polymorphic,or allelic variants. Species variants are isolated using cross-specieshybridization techniques based upon isolation of a full length isolateor fragment from one species as a probe.

Alternatively, antibodies raised against canine RANKL can be used toscreen for cells which express cross-reactive proteins from anappropriate, e.g., cDNA library. The purified protein or definedpeptides are useful for generating antibodies by standard methods, asdescribed above. Synthetic peptides or purified protein are presented toan immune system to generate monoclonal or polyclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. The resulting antibodies are used, e.g., for screening,panning, or sorting.

Example 4 Preparation of Rat Anti-Canine RANKL mAb

Rat anti-canine RANKL mAbs are produced from splenocytes of an 8 weekold female Lewis rat (Harlan Sprague-Dawley, Indianapolis, Ind.)immunized with canine RANKL.Ig fusion protein. The rat is primed i.p.with 25 μg of fusion protein in complete Freund's adjuvant andsubsequently boosted three times i.p. with 10 μg (day 25), 5 μg (day 40)and 10 μg (day 54) in incomplete Freund's adjuvant, respectively. Thefinal boosts are performed both i.v. and i.p. at day 83 with 10 μg offusion protein in saline solution and incomplete Freund's adjuvant,respectively. Splenocytes are fused at day 87 with mouse myelomaP3X63-AG8.653 using PEG 1500 (Roche Diagnostics, Mannheim, Germany).Hybridoma supernatants are screened by indirect ELISA, on both thefusion and control Ig protein, to identify specific mAb-producinghybridomas. These are further characterized by methods such as Westernblot, immuno-precipitation and FACS analysis (e.g., on canine activatedT cells). Selected positive hybridoma lines are subcloned and grown inserum free medium supplemented with SITE (Sigma, St. Louis, Mo.).Antibodies are purified via HiTrap SP and Q columns (Amersham PharmaciaBiotech), and screened for their ability to inhibit RANKL-inducedbiological responses such as activation of NF-κB or activation ofosteoclasts using, e.g., the OCL formation assay described by Yasuda etal., Osteoclast differentiation factor is a ligand forosteoprotegerin/osteoclastogenesis-inhibitory factor and is identical toTRANCE/RANKL, Proc. Natl. Acad. Sci., vol. 95, pp. 3597-3602 (1998).

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the present invention is to belimited only by the terms of the appended claims, together with the fullscope of equivalents to which such claims are entitled. Numerousreferences are cited in the specification, the disclosures of which areincorporated by reference in their entireties.

1. An isolated nucleic acid molecule encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO:2.
 2. The isolated nucleic acidmolecule of claim 1 that comprises the nucleotide sequence of SEQ IDNO:1.
 3. A replicable nucleic acid vector comprising the nucleic acidmolecule of claim
 2. 4. A host cell comprising the replicable nucleicacid vector of claim
 3. 5. A nucleic acid molecule that is a complementto said isolated nucleic acid molecule of claim 1; wherein saidcomplement is the complementary strand to said isolated nucleic acidmolecule.
 6. The isolated nucleic acid molecule of claim 1 that is DNAor RNA.
 7. A replicable nucleic acid vector comprising the nucleic acidmolecule of claim
 1. 8. A host cell comprising the replicable nucleicacid vector of claim
 7. 9. A polynucleotide that hybridizes understringent conditions to the complement of an isolated nucleic acidmolecule encoding a polypeptide comprising the amino acid sequence ofSEQ ID NO:2; provided that said polynucleotide does not encode a human,a murine, or a rat receptor activator of NF-κB ligand polypeptide;wherein said stringent conditions include a hybridization temperature at65° C., in 0.5M Na₂HP0₄, 7% SDS, and 0.5 mM EDTA, pH 8.0, and washes at65° C., with an initial wash in 6×SSC, 0.1% SDS, for 15 minutes,followed by two subsequent washes in 0.2×SSC, 0.1% SDS for 15 minutes.10. A replicable nucleic acid vector comprising the nucleic acidmolecule of claim
 9. 11. A host cell comprising the replicable nucleicacid vector of claim
 10. 12. An isolated nucleic acid moleculecomprising an open reading frame encoding an immunogenic fusion proteincomprising a canine receptor activator of the NF-κB ligand comprisingthe amino acid sequence of SEQ ID NO:2 joined to one or more additionalpeptide domains.
 13. The nucleic acid molecule of claim 12 that is RNAor DNA.
 14. A replicable nucleic acid vector comprising the nucleic acidmolecule of claim
 12. 15. The replicable nucleic vector of claim 14selected from the group consisting of a plasmid, a phage, a cosmid, amini-chromosome, and a virus.
 16. The replicable nucleic vector of claim14 that is suitable for expression of the vector by a eukaryotic hostcell, a prokaryotic host cell, or both.
 17. The replicable nucleicvector of claim 14, comprising a suitable promoter operably linked 5′ tothe open reading frame encoding said immunogenic fusion protein.
 18. Thereplicable nucleic vector of claim 17 that comprises an operably linkednucleic acid sequence encoding a leader peptide enabling secretion ormembrane integration of said immunogenic fusion protein.
 19. A host cellcomprising the replicable nucleic acid vector of claim
 14. 20. The hostcell of claim 19 that is a microorganism selected from the groupconsisting of a bacterium, a yeast, and a protozoan.
 21. The host cellof claim 19 that is derived from a multicellular organism selected froma fungus, an insect cell, a plant cell, and a mammalian cell.
 22. Amethod of producing an immunogenic fusion protein comprising culturingthe host cell of claim 19 under conditions suitable for expressing saidimmunogenic fusion protein.